Determination of Tanshinone IIA in two kinds of Chinese traditional patent medicines by HPLC

Abstract A HPLC method for the determination of Tanshinone IIA in the Dan Qi Pian and Tong Mai Chongji was established. The sample was dissolved in methanol and extracted by ultrasound, then chromatographed on LUNA-C18 column with mobile phase of methanol-water (84:16). The flow rate was set at 0.8mL/min and the detection wavelenghth was of 270nm. This HPLC method is simple, rapid and accurate, it can be used to determine Tanshinone IIA in the Dan Qi Pian and Tong Mai Chongji quantitatively .
Keywords Tanshinone IIA, Dan Qi Pian, Tong Mai Chongji, HPLC

Dan Qi Pian and Tong Mai Chongji are general Chinese traditional patent medicines. Although they are edited in the eighth volume(1993) of Medicine Standards published by the Ministry of Health of the Peoples Republic of China, there was not any determination method for their effective components. Hence it was difficult to examine the quality of the drugs. It is showed that Tanshinone IIA is a kind of important effective component in Dan Qi Pian and Tong Mai Chongji[1]. The determination of Tanshinone IIA have already been reported [2,3]. As complicated components existing, determination method for Tanshinone IIA in Dan Qi Pian and Tong Mai Chongji has not been reported. In this paper, a HPLC method for the determination of Tanshinone IIA in Dan Qi Pian and Tong Mai Chongji was established.

1 INSTRUMENT AND REAGENTS
LC-6A liquid pump of Shimadzu Corp. of Japan, SIL-6B automatic sample injector, SPD-6AV ultravoilet-visible spectrophotometric detector and Chromatopac C-R 6A data processor. Standard sample of Tanshinone IIA was purchased from the Medicine and Biological Products Identification Institute of China. Dan Qi Pian and Tong Mai Chongji were self-made.(according to the eighth volume(1993) of Medicine Standards published by the Ministry of Health of the Peoples Republic of China). Methanol was HPLC grade.

2 METHOD AND RESULTS
2.1 Chromatographic conditions

LUNA C18 column (4.6mm ID×250mm, particle diameter 5mm) was used. The column temperature was at 35°C. The mobile phase was methanol-water (84:16) and the flow rate was set at 0.8mL/min. The detection wavelength was of 270nm, the recorder paper rate was at 1mm/min.
2.2 Experiment operation
The standard sample was diluted to suitable concentration by methanol, then injected. The negative samples without Tanshinone IIA were prepared respectively according to Dan Qi Pian and Tong Mai Chongji prescription. The samples of Dan Qi Pian and Tong Mai Chongji and their negative samples were taken and extracted according to the sample determination method, injected and the chromatograms were given in Figure 1 and 2. From the chromatograms, it was known that the other components of Dan Qi Pian and Tong Mai Chongji had not affected the determination of Tanshinone IIA. The number of theoretical plates was 22339 with regard to the peak of Tanshinone IIA in Dan Qi Pian, and the resolution between Tanshinone IIA and its neighbouring peak was 3.3. The number of theoretical plates was 22000 with regard to the peak of Tanshinone IIA in Tong Mai Chongji, and the resolution was 3.0. Under the chromatographic conditions we selected, Tanshinone IIA in Dan Qi Pian and Tong Mai Chongji can be determined quantitatively.

Fig.1 The chromatogram of (A) Tanshinone IIA (B) negative sample of Dan Qipian (C) Dan Qipian  the peak of 1 in chromatogram (C) is Tanshinone IIA

Fig.2 The chromatogram of (A) Tanshinone IIA (B) negative sample of   Tong Mai Chongji (C) Tong Mai Chongji  the peak of 1 in chromatogram (C) is Tanshinone IIA
2.3 Linear range
The standard sample was taken precisely and diluted to five grade concentration with methanol, then injected 10 mL respectively and the peak area was recorded. The linear regression was made between the mean peak area of Tanshinone IIA X and the injected concentration Y(mg/mL), and r was 0.9999. The linear range was 4.14~62.10 mg/mL.
2.4 Apparatus precision and method repeatability
A sample solution was taken into the automatic sample injector, and injections were made in 5h for n=5, interday RSD% of Dan Qi Pian and Tong Mai Chongji was both 0.5. The same sample was determined for three consecutive days and five times per day (n=5), intraday RSD% of Dan Qi Pian and Tong Mai Chongji was 4.2 and 3.9 respectively.
2.5 Adding standard recovery
Six parts of the same batch Dan Qi Pian and Tong Mai Chongji were weighed precisely respectively, Tanshinone IIA standard sample was added precisely into five parts, the other part was the blank. Every part was operated according to the sample determination method, for the determination of the Tanshinone IIA concentration. The average recovery of Dan Qi Pian and Tong Mai Chongji was 100.7% and 100.4% respectively, and RSD% was 1.8 and 2.2 respectively.
2.6 Sample determination
2.6.1 Dan Qi Pian
Twenty Dan Qi Pians were taken and weighed precisely, the average weight of the tabelet was calculated, then ground into powder. A certain amount of the powder (about weight of ten tabelets Dan Qi Pian ) was taken and weighed precisely to a 25mL volumetric flask, then a certain amount of methanol was added. Extracted by ultrasound for 5 minutes, and supplemented the lost methanol to the calibration tails, then filtered. An aliquot of 10mL was injected , the peaks of Tanshinone IIA was recorded and its amount was calculated by the external standard method. The average concentration of Tanshinone IIA in Dan Qi Pian was 0.018mg/g, RSD was 0.7%(n=5).
2.6.2 Tong Mai Chongji
A certain amount(about 5 g) of Tong Mai Chongji was precisely weighed and taken into a 25mL volumetric flask. A certain amount of methanol was added. Extracted by ultrasound for 5 minutes, and supplemented the lost mathanol to the calibration tails, then filtered. An aliquot of 10mL was injected , the peaks of Tanshinone IIA was recorded and its amount was calculated by the external standard method. The average concentration of Tanshinone IIA in Tong Mai Chongji was 0.075mg/g, RSD was 0.7%(n=5).

3 CONCLUSION
Under the chromatographic conditions selected by us, the HPLC method we established can be used to determine Tanshinone IIA in Dan Qi Pian and Tong Mai Chongji quantitatively. The method was sensitive, rapid and accurate.

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